SOLUTION DE FIXATION UTILISABLE EN VUE DE LA FIXATION ET DE LA PRESERVATION D'ECHANTILLONS BIOLOGIQUES

FIXATIVE SOLUTION, FOR FIXATION AND PRESERVATION OF BIOLOGICAL SAMPLES

Abrégé français

La présente invention concerne une solution de fixation pouvant être utilisée en vue de la fixation de substances biologiques telles que des cellules, des organes, des sécrétions et des tissus biologiques humains ou vétérinaires, ainsi que pour des bactéries, des virus, des levures, des parasites et des produits biotechnologiques comme des articles d'embaumement. Le caractère inédit de cette invention tient à ce que cette solution de fixation ne constitue pas un produit dangereux au sens des normes européennes. Elle contient une faible concentration en aldéhydes ou en métaux lourds et donne un produit qui ne peut être considéré comme dangereux. Cela n'empêche pas cette solution de permettre une préservation et une fixation optimales des échantillons biologiques.

Abrégé anglais

The invention refers to a fixative solution, suitable for fixation of
biological materials such as human or veterinary
biological tissues, cells, organs and secretions as well as bacteria, viruses,
yeasts, parasites and biotech products such as embalming
articles. New in this invention is the property of this fixative solution not
to be a dangerous product towards European standards, it
contains low concentration of aldehydes or heavy metals, rendering a product
that cannot be considered dangerous. Even so, biological
samples can be optimally preserved and fixed with this solution.

Revendications

13
THE EMBODIMENTS OF THE PRESENT INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method comprising: fixing and preserving biological
materials by placing said materials in a composition comprising
halocyanoacetamide and a pH adjuster selected from the group
consisting of acetic acid, phosphoric acid, citric acid, and
formic acid.
2. The method according to claim 1, wherein the
halocyanoacetamide is 2,2-dibromo-2-cyanoacetamide.
3. The method according to claim 1 or 2, wherein the
composition additionally comprises a solvent selected from
water, organic solvents and mixtures thereof.
4. The method according to any one of claims 1 to 3, wherein
the composition has a pH adjusted to a range between pH 3.0
and pH 6Ø
5. The method according to any one of claims 1 to 4, wherein
the composition additionally comprises at least one of a
humectant; a surfactant; a salt of alkali metals or alkaline
earth metals; an antioxidant; and dyes.
6. The method according to claim 5, wherein the humectant
is selected from the group consisting of ethylene glycol,
propylene glycol, glycerine, erithriol, sorbitol, andmixtures
thereof.
7. The method according to claim 5, wherein the surfactant

14
is selected from the group consisting of non-ionic surfactants,
sodium dodecyl sulphate and mixtures thereof.
8. The method according to claim 5, wherein the alkali metals
and alkaline earth metal salts are selected from the group
consisting of citrates, acetates, phosphates, chlorides,
sulphates, formates, and nitrates of lithium, sodium,
potassium, cesium, magnesium, calcium, strontium, barium, and
mixtures thereof.
9. The method according to claim 5, wherein the antioxidant
is selected from the group consisting of
4,4-bis(2,6-di-tert-butylphenol)4,
4-bis(2,6-di-tert-buthyl-p-cresol), and mixtures thereof.
10. The method according to claim 5, wherein the composition
comprises: 0.05%-5% 2,2-dibromo-2-cyano acetamide, citric
acid, 1%-30% propylene glycol, 0 . 1%-5 % non-ionic surfactants,
0.001%-0.1% dye, 0.05%-0.1% antioxidant 4,4-bis
(2,6-di-tert-butylphenol), and 0.01-10% sodium chloride.
11. The method according to claim 5, wherein the composition
comprises: 1% 2,2-dibromo-2-cyanoacetamide, 0.5% citric acid,
12% propylene glycol, 0.1% non-ionic surfactants, 0.05%
aromatic oils, 0.05%
antioxidant
4,4-bis(2,6-di-tert-butylphenol), 0.01% dye, and 0.1% sodium
chloride.
12. The method according to anyone of claims 1 to 11, wherein
the composition is a powder, tablet, capsule, gel, or varnish.

Description

1
FIXATIVE SOLUTION, FOR FIXATION AND PRESERVATION
OF BIOLOGICAL SAMPLES
FIELD OF THE INVENTION
The invention refers to a fixative solution, suitable
for fixation of biological materials such as human or
veterinary biological tissues, cells, organs and secretions
as well as bacteria, viruses, yeasts, parasites and biotech
products such as embalming articles.
BACKGROUND OF THE INVENTION
Typical for formaldehyde, chromic acid, osmic acid and
other components attachable, is the irreversible denaturation
of proteins, which has a decisive influence on the protein
composition of the sample.
In the biochemical determination of enzymes, proteins
and immunological and other protein-specific examinations,
these conventional fixative substances cannot be used.
Presently, formaldehyde (formalin) is the most used
fixative substance, which has excellent fixative properties
even for longer periods of time.
Aldehydes, especially formaldehyde, are considered
highly toxic.
It was classified as a justified potential carcinogen
chemical and it can cause allergies and dermal, respiratory
and eye irritations. In case of exposure to high doses, it can
even result in death risk. It also has a characteristic pungent
CA 2834817 2017-09-19

2
and unpleasant odor.
For reasons of environmental protection and work safety,
the use of formalin is legally restricted.
Fixative solutions without formaldehyde are known from
patents EP 1 455 174 El, U.S. Pat. No. 6,531,317B2, WO 03/029
783 Al, DE 699 17 779 T2, U.S. Pat. No. 5,422,277A, US
2005/0084924 Al and DE 26 30 823 Al.
There are several ideas to create a festive solution
without formaldehyde, such as described in DE 38 22 183 Al,
where Tannic Acid is suggested as the main component of the
solution. To obtain a quick fixation the patent DE 38 24 936
Al suggests the utilization of hot water action, with the aid
of Tannic Add and a Bi-alcohol in the water. The patent DE 44
04 544 Al rejects entirely chemical means of fixation, such
as formalin, suggesting a denaturation of proteins from the
sample by the action of heat through hot water baths. This
procedure has the disadvantage, however, that the structures
of the material that is being fixed may undergo morphological
changes. Proteins, for example, can clot. For this reason, this
procedure is not suitable for all types of materials.
Other fixative solutions are known in the literature.
Almost all of them, with few exceptions, contain very dangerous
substances and are a great potential hazard to living beings
and the environment.
Among the best known, we can include:
Schaudinn's Fixative (it contains mercury salts), SAF
(it contains formaldehyde), MIF (it contains formaldehyde and
CA 2834817 2017-09-19

3
mercury salts) , osmic acid (it is carcinogenic, toxic, caustic),
chromic acid, (carcinogenic, toxic, caustic), FA (formaldehyde
and alcohol) and others which are mentioned in the literature.
In general, a fixative solution must maintain biological
materials such as biological tissues, cells, clustering of
cells, viruses, bacteria, parasites, yeasts, feces and other
in a stable state for a relatively long period of time after
taking the sample. The original state of the sample immediately
after collection should be fixed for a further examination in
the laboratory (e.g., a microscopic, examination) so
morphological state present at the collection of the sample
can be found. Importantly, the morphological structure of the
sample must be preserved. There shall be no chemical, physical
or microbiological processes or proliferation of bacteria,
yeast or fermentation or decomposition processes or other
degradation processes, which may adversely affect the sample.
In short, the fixative solution should keep any type of
biological material in a stable state suitable for preparation
or analytical purposes.
The ideas previously described which are in the
current-state of the art, are oriented Lo avoid completely the
use of formalin, replacing it by nontoxic substance or less
hazardous ones, although they show as much as possible the same
characteristics that formalin fixation does.
The fixation is based on different chemical mechanisms,
some of which are not yet fully elucidated. One major mechanism
is the so-celled "Crossing Over". In this process, there is
CA 2834817 2017-09-19

4
a chemical bond of the molecule in the fixative which can be
covalent type, Van-der-Waals interaction or even ionic to a
corresponding position in a protein molecule in a sample. It
is possible that there are different types of bonding at the
same time. With the raise of chemical bondings in biological
material. there is an increase in the mechanical stability of
biological structures and at the same time microorganisms in
the sample are destroyed.
Until now, fixative solutions were designed to maximize
these processes. With this, there is always an excess of
fixative substance in the solution, which is not always
necessary or even convenient. (If there is excess of toxic
substances in the workplace, there will be excessive reaction
with the sample.)
SUMMARY OF THE INVENTION
In an aspect of the invention, this fixative
solution is not a dangerous product by European standards. It
contains low concentration of aldehydes or heavy metals,
rendering a product that cannoL be considered dangerous. Even
so, biological samples can be optimally preserved and fixed
with this solution.
An aspect of the presenL invention creates a fixative
solution which is not dangerous, and has the qualities of
formalin in milder form. A composition with other fixative
substances should be possible to an adjustment of the ideal
fixation process.
CA 2834817 2017-09-19

=
Another advantage of an aspect of the invention is that
there is not such a strong reaction between the sample and the
new fixing solution. With this, the sample is set intact and
fixed until further processing, and proteins and other
5 components from the sample are not strongly altered by the
solution, enabling the detection of biochemical factors that
would be destroyed in a conventional fixative solution.
According to an aspect of the present invention, there
is provided a method comprising: fixing and preserving
biological materials by placing said materials in a composition
comprising halocyanoacetamide and a pH adjuster selected from
the group consisting of acetic acid, phosphoric acid, citric
acid, and formic acid.
DETAILED DESCRIPTION OF THE INVENTION
Aspects of the invention consist of providing a fixative
solution that:
- Is in accordance with valid rules and (in this case
rules of the European community) the solution is not
dangerous or just little dangerous to the user and the
environment. (In any case less dangerous than the
traditional fixation solutions).
- Does not present other forms of danger (flammable,
explosive, corrosive, accumulating in the environment
or highly volatile).
- Reacts in a mild way with the sample and the sample
proteins, causing no strain on the morphological
CA 2834817 2017-09-19

6
structure of the sample ("shrinkage") and keeps intact
more biological and biochemical parameters than
traditional solutions.
- Does not significantly disturb the processes in which
the sample has to undergo after fixation (such as
unwanted reactions, preventing staining and others).
Two basic substances were chosen to produce the desired
effect:
- Halogen cyano-acetamides
- 2,2-dibromo-2-cyano Acetamide or
2,2-Dibromo-3-nitrilopropionamide or for short
DBNPA.
In order to simplify, we will call the mentioned substance
as DBNPA.
These substances were chosen because of the following:
Halogen cyano-acetamides and DBNPA have strong biocidal
properties and completely interrupt the biological and
microbiological activities in the sample.
Because of the molecular structure of these substances,
the effect of crossing over can occur because these halogen
atoms produce molecular polarization needed for the occurrence
of van-der-Waals interactions between the molecule of fixative
and protein molecules of the sample, thereby ensuring
stabilization of the structure and thus fixation. Due to high
biocidal properties of the substance, minimum concentration
may be applied, avoiding unnecessary excesses. The biological
activity of the sample ceases quickly, avoiding the
CA 2834817 2017-09-19

7
microbiological degeneration of the sample.
The optimal action of the solution occurs at acid pH or
around the neutral point, but depending on other components
of the solution and the desired results, it can be required
to adjust the pH to a range from 3-9.
The fixative solution of the present invention can be
prepared in liquid form. However, it is also possible to present
it in a concentrated form which provides a turnkey solution,
after addition of an appropriate solvent, which in the simplest
case is water. This (concentrate) can be a concentrated
solution, a powder mixture, a soluble gel, a tablet, a soluble
capsule, a soluble varnish covering the container with the
solution or another similar form.
With this transportation, storage and packaging costs
can be reduced, besides other advantages of economic order and
logistics as the solvent, water in most cases, can be added
at the final destination by the user.
A concrete example is a solution using 2% by weight DBNPA,
and other substances as an additional acid to adjust pH to
slightly acidic. Organic or inorganic acids can be utilized
such as citric acid. A moisturizer can be added, such as Ethylene
Glycol, Glycerin, Propylene Glycol, Sorbitol, Erythriol or
similar, which also serve as mediators to the lipid phase of
the sample, facilitating its spread. Another component to be
added would be an ionic or anionic surfactant, or a mixture
of both, in order to lower the surface tension of the solution,
improving the dispersive features. From these substances we
CA 2834817 2017-09-19

8
can list ethoxylated polyethylene glycol, ethoxylated sorbates
(commercially TWEEN) , sodium dodecyl sulfate, sodium benzene
sulphonate and other Alkyl sulfates and sulphonates, as well
as commercial products in the series TRITON, ETHOMEEN and other
similar ones. Fatty alcohols (N>8) such as mono or polyalcohols,
linear or branched, can be applied as process aids and mediators
of the surfactants used.
For improvement of the solution for marketing purposes,
flavorings and colorings that do not conflict with other
components will be added.
For isotonic adjustment purposes, salts of alkali metals
or alkaline earth such as chlorides, sulfates, nitrates,
citrates, acetates of lithium, sodium, potassium, magnesium,
calcium, strontium, or others, can be used.
For pH adjustment purposes in certain applications, it
may be necessary to utilize buffer solutions such as citrate
buffer, acetate or others known in literature.
It is known from literature that halogeno cyano
acetamides, especially DBPNA, undergo alkaline hydrolysis when
in solution. For the solution to guarantee a longer period
operation, small amounts of specific antioxidants should be
added to the solution to avoid an early deterioration. The
solution thus treated, maintains its activity for a period of
about two years. The degradability of the solution when
disposed is not affected, maintaining its ecological feature.
As we can see in the diagram below, the final
decomposition products of DBPNA are not accumulative in the
CA 2834817 2017-09-19

9
environment:
pH 5.4 Hydrolysis Br 0 Nucleophiles
25 I 4/
TH = ¨9 hr NCCC Light
I \õ
NCCHBr3 + CO2 + NH2 Br i xµk-L2
NCCH3CONH2 + 2 Br
pH 7.4 TH = 21 days H2O
[Br2CHCONH2] NCCH2CO2H
H20
Br2 + CHCO211 + NH2
0
pH 7.4 TH= 300 days
CCH2CO2H
0,µ
112N
CCO2H + 2 Br"
fast
1102CCH3CO2H
vl
slow
HO2CCO2H 2CO2
Photodecomposition is delayed with the use of a dye-ink
in the solution, and the recommendations of storage in dark
and fresh place.
The hydrolysis is greatly reduced with the use of
commercial antioxidants such as 4,4-
bis
(2,6-di-tert-butylphenol)or 2,6-di-tert-butyl-p-cresol or
similar ones, which is practiced commercially.
It is important to highlight that formaldehyde as one
of the decomposition products from DBPNA, is inhibited due to
CA 2834817 2017-09-19

10
stabilization by antioxidants. For the users, who need their
biological sample to be fixed for a processing that by rule
it takes only a few weeks the solution does not present levels
of formaldehyde that may affect the mild fixation.
Commercial biocides solutions of DBPNA used in the paper
and textile industry thus stabilized show a degradation of
DBPNA fewer than 10% in 20 months, ensuring an optimal action
of the component.
SIMPLE FORMULATION EXAMPLE:
DBPNA 2%
Propylene Glycol 5%
TWEEN 80 1%
Food coloring 0.01%
Mixture of fragrances 0.01%
Antioxidant 0.05%
Water to make 100%
This solution was tested and evaluated with samples of
human and animal feces from the parasitological point of view;
eggs of parasites were mainly detected. Histological samples
from bovine muscle were also fixed, showing excellent quality
of fixation and preservation. During the 30 days observation
period, no changes were observed in any of the analyzed samples.
In a subsequent evaluation, human feces samples were found
contaminated with protozoa. Protozoa in feces are extremely
susceptible to degradation and can degenerate in a few hours.
CA 2834817 2017-09-19

11
In the tests, preservation and fixation were demonstrated for
a period exceeding seven days. Compared to a standard solution
of formaldehyde, the quality of preservation was equal if not
better.
EXAMPLE OF SOLUBLE TABLET SIMPLE FORMULATION:
DBPNA 100g
Sorbitol powder 41g
TWEEN 80 2.5g
Citric Acid 2.0g
Food coloring 1.0g
Mixture of fragrances 1.0g
Antioxidant 2.5g
Gum arabic 50g
Total Mass 200g
One 40 g-tablet of this mass contains 20 g of DBPNA used
to produce 1 liter of 2% solution ready-to-use
At 2% concentration of DBPNA in the solution should bring
only the risk phrase R43, indicating the risk of sensitization
through skin contact. At concentrations below 1% solution does
not need indication as dangerous product.
It is not flammable, corrosive, does not produce toxic
or dangerous emanations, it is not teratogenic or carcinogenic,
is not offensive to the environment and it requires no special
precautions for work safety, bringing about an extraordinary
improvement in job security for the user as well as a remarkable
CA 2834817 2017-09-19

12
simplification in the processes of environmental management
in the laboratory in which it is being utilized.
It should be stated here that, besides water, other
solvents may be utilized, depending on the desired application.
With advances in diagnostic techniques, alcohol and other
solvents such as methyl, ethyl, propyl, butyl, ketones, esters,
ethers, glycerin and other organics are being discussed. The
fixative described can be obtained with the use of organic
solvents and mixtures of organic solvents or mixtures of
organic solvents with water.
CA 2834817 2017-09-19